Cytotoxic compounds: derivatives of the pyrido [2,3,4-kl]acridine ring system

ABSTRACT

Compounds of formula (I), wherein X is selected from the group consisting of O, and NR 3 , where R 3  represents a lower alkyl group; Y is selected from the group consisting of CH and N; R 1  and R 2  are independently selected from the group consisting of NH 2 , NHR 4  and NR 5   2 , where R 4  and R 5  each represent a lower alkyl group, or R 1  and R 2  together represent a cycle selected from (a), (b) and (c), wherein R 6 , R 7  and R 8  are independently selected from the group consisting of hydrogen atoms, lower alkyl groups, hydroxy groups and lower alkoxy groups; and Z is selected from the group consisting of O.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to a series of new polycyclicaromatic alkaloids having a pyrido[2,3,4-k,l]acridine skeleton whichhave cytotoxic properties and which can therefore be used in thetreatment of malignant tumours. The invention also provides methods andcompositions using these new compounds as well as processes for theirpreparation.

[0002] The polycyclic aromatic alkaloids based on thepyrido[2,3,4-k,l]acridine skeleton are a growing class of ascidianmetabolites that often exhibit a variety of interesting biologicalproperties, including antitumour activity.¹ This class of compoundscomprises three main structural types, depending of the position of thefusion between the parent structure and additional rings present in thenatural product. For example, the cystoditines² have the base skeletonmentioned above, while amphimedine,³ meridine⁴ and cystodamine⁵ bear anadditional pyridine ring attached to the h bond; ascididemin,⁶ itsderivatives,⁷ the kuanoniamines⁸ and shermilamine A⁹ show thisadditional ring at the i face, and eilatine at both.¹⁰ Our targetcompounds can be regarded as regioisomers both of meridine andamphimedine, but they have not been so far isolated from naturalsources.

[0003] Most of these prior art compounds exhibit very interestingcytotoxic properties towards a range of tumor cell lines. Although theirmechanism of action is not clearly established, three generalobservations emerge from published biological data:¹¹ a) they areintercalating agents; b) they disrupt DNA and RNA synthesis, with littleeffect on protein synthesis; c) they inhibit topoisomerase II, which isthe mechanism normally accepted for their antitumour activity.

[0004] Biological studies on pyridoacridines are severely limited due totheir very low availability from natural sources, and therefore thestudy of their mechanism of action and the establishment of reliablestructure-activity relationships requires the development of efficientsynthetic routes.

[0005] Definition of the Invention

[0006] We have now discovered a new family of polycyclic aromaticalkaloids having a pyrido[2,3,4-k,l]lacridine skeleton which showexcellent antitumour activity.

[0007] Thus, in a first aspect of the present invention there isprovided compounds having the general formula (I):

[0008] wherein:

[0009] X is selected from the group consisting of O, and NR³, where R³represents a lower alkyl group;

[0010] Y is selected from the group consisting of CH and N;

[0011] R¹ and R² are independently selected from the group consisting ofNH₂, NHR⁴ and NR⁵ ₂, where R⁴ and R⁵ each represent a lower alkyl group,or R¹ and R² together represent a cycle selected from (a), (b) and (c):

[0012] wherein R⁶, R⁷ and R⁸ are independently selected from the groupconsisting of hydrogen atoms, lower alkyl groups, hydroxy groups andlower alkoxy groups; and

[0013] Z is selected from the group consisting of O and NH;

[0014] and pharmaceutically acceptable salts thereof.

[0015] In the definitions of the groups in formula (I), the lower alkylgroups and the lower alkyl moiety of the lower alkoxy groups arestraight-chain or branched alkyl groups having 1 to 6 carbon atoms suchas methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl,tert-butyl, pentyl, neopentyl and hexyl groups.

[0016] For those compounds of the present invention wherein R¹ and R²together represent a cycle of formula (a), R⁶ and R⁷ are preferablyindependently selected from the group consisting of hydrogen atoms,alkyl groups having from 1 to 4 carbon atoms, hydroxy groups and alkoxygroups having from 1 to 4 carbon atoms; more preferably, R⁶ and R⁷ areindependently selected from the group consisting of hydrogen atoms,methyl groups and ethyl groups; and, most preferably, R⁶ represents amethyl group and R⁷ represents a hydrogen atom. Furthermore, where R¹and R² together represent a cycle of formula (a), it is preferable thatZ represents a group of formula NH.

[0017] For those compounds of the present invention wherein R¹ and R²together represent a cycle of formula (b) or (c), R⁶, R⁷ and R⁸ arepreferably selected from the group consisting of hydrogen atoms, alkylgroups having from 1 to 4 carbon atoms, hydroxy groups and alkoxy groupshaving from 1 to 4 carbon atoms; more preferably, R⁶, R⁷ and R⁸ areindependently selected from the group consisting of hydrogen atoms,methyl groups, ethyl groups and hydroxy groups; and, most preferably, R⁶represents a hydroxy group and R⁷ and R⁸ each represent a hydrogen atom.

[0018] In a preferred embodiment, the present invention relates to novelsynthetic compounds of general structure (II) or (III):

[0019] wherein R⁶, R⁷ and R⁸ are as defined above.

[0020] Of the compounds of general formula (II), we particularly preferthe compound wherein R⁶ represents a methyl group and R⁷ represents ahydrogen atom (Compound No. IB-96213).

[0021] Of the compounds of general formula (III), we particularly preferthe compound wherein R⁷ and R⁸ both represent a hydrogen atom (CompoundNo. IB-98205).

[0022] The present invention also provides a method for treating amammal affected by a malignant tumor sensitive to a compound having thegeneral formula (I), which comprises administering to the affectedindividual a therapeutically effective amount of the compound having thegeneral formula (I) or a pharmaceutical composition thereof.

[0023] The present invention further provides pharmaceuticalcompositions, particularly useful in the treatment of malignant tumors,which contain as the active ingredient a compound having the generalformula (I), as well as a process for the preparation of saidcompositions.

[0024] A further aspect of the present invention provides a method forpreparing the compounds of general formula (I) and, in particular,Compounds Nos. IB-96213 and IB-98205.

[0025] Our strategy for the synthesis of the target compounds involvesan hetero Diels-Alder reaction between an o-nitrogenated4-aryl-1-dimethylamino-1,4-azadiene and a suitable heterocyclic quinone,followed by nucleophilic cyclization onto one of the quinone carbonyls(Scheme 1).

[0026] Among the several 4-aryl-l-azadienes assayed, the best resultswere obtained for the o-nitro and the o-(trifluoroacetamido)derivatives. The first of them (compound 1a) was prepared fromcommercially available o-nitrocinnamaldehyde using a known procedure¹².For the second azadiene (compound 1b), two alternative syntheses weredevised (Scheme 2). The first of them involved trifluoroacetylation ofthe corresponding o-amino derivative 2, prepared by reduction ofcompound 1a¹². In the second synthesis, o-aminobenzaldehyde (3) wasN-trifluoroacetylated and the amide was used as a Wadsworth-Emmonscoupling partner with compound 5.¹³

[0027] The reaction between 1b and quinones 6¹⁴ to produce compounds offormula (II) was carried out in refluxing chloroform, and led to amixture of the Diels-Alder adducts 7 and the secondary products 8.Compounds 7 were aromatized to 9 by refluxing in the presence of Pd-C inmethanol solution, and 9 was finally transformed into the desiredpentacyclic derivatives 10 [general structure (II)] by hydrolyticcyclization (Scheme 3).

[0028] The use of 6-bromo-4-chloroquinolinequinones 11 as dienophilesled to reversal of the regioselectivity of the hetero Diels-Alderreaction. Thus, treatment of azadiene 1a with 11 under ultrasoundirradiation afforded compound 12. Hydrogenation of 12 in the presence ofPd-C followed by workup with trifluoroacetic acid gave pyridoacridines13 [general structure (III)] (Scheme 4).

[0029] As examples, the detailed procedure for the synthesis of12-methyl-9H-benzo[b]pyrido[4,3,2-d,e](1,7)phenantroline-8,10-dione,IB-96213 (general structure 10, where R⁶=CH₃, R⁷=H) and9-hydroxybenzo[b]pyrido[4,3,2-d,e](1,10)phenantrolin-8-one IB-98205(general structure 13, where R⁷=R⁸=H) are given in the experimentalsection.

[0030] The compounds of the present invention are cytotoxic, compoundssuch as IB-96213 and IB-98205 exhibiting antitumor activity, especiallyagainst cell lines derived from human solid tumors, such as human lungcarcinoma, human colon carcinoma and human melanoma, and, the like. Theyare also active against other tumor cell lines, like leukemia andlymphoma. Compounds of formula (I), such as IB-96213 and IB-98205, havein vitro antitumor selectivity for solid tumors.

[0031] Examples of pharmaceutical compositions include any solid(tablets, pills, capsules, granules, etc.) or liquid (solutions,suspensions or emulsions) dosage form, with suitable formulation oforal, topical or parenteral administration, and they may contain thepure compound or in combination with any carrier or otherpharmacologically active compounds. These compositions may need to besterile when administered parenterally.

[0032] The correct dosage of a pharmaceutical composition comprisingcompounds with formula (1), will vary according to the pharmaceuticalformulation, the mode of application, and the particular situs, host andtumor being treated. Other factors like age, body weight, sex, diet,time of administration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severity of the disease shallbe taken into account. Administration can be carried out continuously orperiodically within the maximum tolerated dose.

Experimental

[0033] The invention is further illustrated by the following Examples,which demonstrate the preparation of various of the compounds of thepresent invention. The reagents used were of commercial origin (Aldrich,Fluka) and were employed without further purification. Solvents (SDS,Scharlau) were purified and dried by standard procedures. Reactions weremonitored by thin-layer chromatography, using Scharlau andMacherey-Nagel plates with fluorescent indicator. Separations by flashliquid chromatography were performed using silica gel SDS 60 ACC(230-400 mesh).

[0034] Melting points are uncorrected, and were determined in opencapillary tubes, using a Büchi immersion apparatus or a Hoffler hotstage microscope. Combustion elemental analyses were obtained by theServicio de Microanalisis Elemental, Universidad Complutense, using aPerkin Elmer 2400 CHN analyzer. Spectroscopic data were obtained withthe following instruments: IR, Perkin Elmer 577 and Perki Elmer Paragon1000 FT-IR; NMR, Varian VXR-300 (300 MHz for ¹H and 75 MHz for ¹³C) andBruker AC-250 (250 MHz for ¹H and 63 MHz for ¹³C).

EXAMPLE 1

[0035]12-Methyl-9H-benzo[b]pyrido[4,3,2-d,e](1,7)phenantroline-8,10-dione(IB-96213; a compound of formula 10 in which R⁶=CH₃ and R²=H)

[0036] Step 1(a)4-(o-Trifluoroacetamidophenyl)-1-dimethylamino-1-azadiene (Formula 1b)

[0037] Method A

[0038] Trifluoroacetic anhydride (1.39 g, 6.63 mmol) was added dropwiseto a stirred solution of 4-(o-aminophenyl)-1-dimethylamino-1-azadiene(2)¹² (837 mg, 4.42 mmol) in dry ethyl ether (10 ml). The solution wasstirred at room temperature for 15 min and evaporated under reducedpressure at room temperature. The residue was purified by chromatographyon silica gel, eluting with dichloromethane to give the title compound1b; yield, 1.023 g (88%). Data for 1b: Mp, 110° C. Analysis. Calcd. forC₁₃H₁₄F₃N₃: C, 54.73: H, 4.91; N, 14.73 Found: C, 54.10; H, 4.19; N,14.11 IR (KBr): 3250 (NH), 1655 (CO). ¹H-NMR (250 MHz, CDCl₃) d: 8.01(1H, s, NHCOCF₃); 7.69 (1H, m, C_(3′)-H); 7.52 (1H, m, C₆-H); 7.25 (2H,m, C_(4,5)-H); 7.08 (1H, d, J = 8.9 Hz, C_(2′)-H); 6.88 (1H, dd, J =15.6 and 8.9 Hz, C_(3′)-H); 6.50 (1H, d, J = 15.6 Hz, C_(4′)-H); 2.88(6H, s, CH₃). ¹³C-NMR (63 MHz, CDCl₃) d: 155.14 (d, J = 37.1 Hz, CO);133.43 (C_(2′)); 1313.84 (C_(3′)); 128.00 (C₄); 127.54 (C₅); 126.30(C₆); 124.61 (C₃); 123.25 (C_(4′)); 115.66 (q, J = 288.0 Hz, CF₃); 42.39(NMe₂).

[0039] Method B.

[0040] a) o-(trifluoroacetamido)benzaldehyde (4). To a suspension ofo-nitrobenzaldehyde (3 g, 19.86 mmol) in 35% aqueous hydrochloric acid(35 ml) was added 21 g (93.1 mmol) of tin (II) chloride in smallportions. The suspension was stirred at room temperature for 72 h,neutralized with 6N aqueous sodium hydroxide and extracted withchloroform (4×50 ml). The combined chloroform layers were dried oversodium sulphate and evaporated, yielding 1.87 g (78%) ofo-aminobenzaldehyde 3. A part of this residue (1.26 g, 10.4 mmol) wasdissolved in dry ethyl ether (5 ml). This solution was cooled to 0° C.and treated dropwise with trifluoroacetic anhydride (2 ml, 14.15 mmol),while magnetically stirred. The solution was stirred for 2 h at roomtemperature. The solvent was evaporated and the residue waschromatographed on silica gel, eluting with a 7:3 petroleumether-dichloromethane mixture. Yield, 1.65 g (77%) of compound 4. Datafor 4: Mp, 71-72° C. Analysis. Calcd. for C₉H₆F₃NO₂: C, 49.78: H, 2.79;N, 6.45 Found: C, 49.70; H, 2.59; N, 6.11 ¹H-NMR (250 MHz, CDCl₃) d:10.21 (1H, s, NHCOCF₃); 9.99 (1H, m, C_(3′)-H); 8.69 (1H, d, J = 8.4 Hz,C₃-H); 7.81 (1H, dd, J = 7.6 and 5.6 Hz, C₆-H); 7.72 (1H, td, J = 7.9and 1.6 Hz, C₅-H); 7.41 (1H, t, J = 7.4 Hz, C₄-H). ¹³C-NMR (63 MHz,CDCl₃) d: 195.62 (CO); 155.70 (d, J = 38.1 Hz, CO); 138.05 (C₁); 136.32(C₄); 136.04 (C₅); 125.22 (C₆); 122.47 (C₂); 120.32 (C₃); 115.44 (q, J =289.2 Hz, CF₃).

[0041] b) Wadsworth-Emmons reaction between compounds 4 and 5. To astirred solution of hydrazone 5¹³ (see Scheme 2) in dry tetrahydrofuranplaced in a −78° C. bath was added a solution of butyl lithium inhexanes (0.1 ml, 1.1 mmol). The coloured solution was stirred for 40 minat the same temperature and a solution of the aldehyde 4 (217 mg, 1mmol) in dry tetrahydrofuran (1 ml) was then added. The reacting mixturewas stirred at 0° C. for 4 h, quenched by addition of a saturatedaqueous solution of ammonium chloride (10 ml) and extracted withchloroform (4×15 ml). The combined extracts were dried over sodiumsulphate and evaporated and the residue was chromatographed on silicagel, eluting with 1:1 ethyl ether-petroleum ether. Yield, 103 mg (36%)of the title compound 1b.

[0042] Step 1(b)5-(o-Trifluoroacetamido)-4methyl-5,8-dihydro-1,8-diazaanthracene-2,9,10-trione(a compound of formula 7 in which R⁶=CH₃ and R⁷=H)

[0043] A solution of quinone 6 (see Scheme 3; R⁶=CH₃, R⁷=H)^(14a) andazadiene 1b produced according to Step 1(a) above (32 mg, 0.17 mmol) inchloroform (50 ml) was refluxed in a bath at 60° C. for 22 h, while astream of argon was forced through the solution. The reaction mixturewas evaporated to dryness and the residue was purified by chromatographyon silica gel, eluting with a gradient from 3:2 dichloromethane-ethylacetate to neat ethyl acetate, yielding 25 mg (34%) of the title5,8-dihydro derivative 7 (R⁶=CH₃, R⁷=H), 20 mg (28%) of the aromaticcompound 9 (see Scheme 3; R⁶=CH₃, R⁷=H) and 11 mg (28%) of compound 8(see Scheme 3; R⁶=CH₃, R⁷=H).^(14a) Data for 7: Mp 192° C. (AcOEt)Analysis. Calcd. for C₂₁H₁₄F₃N₃O₄: C, 58.77: H, 3.36; N, 9.78 Found: C,58.49; H, 3.62; N, 9.57 IR (KBr): 3364 (NH); 1726, 1661 (CO) cm⁻¹.¹H-NMR (250 MHz, CDCl₃) d: 11.93 (1H, br, s, NH); 11.28 (1H, s,NHCOCF₃); 9.30 (1H, br, s, NH) 7.37-7.32 (4H, m, C_(2′-6′)-H); 6.50 (1H,s, C₃-H); 6.35 (1H, m, C₇-H); 4.96 (1H, m, C₆-H); 4.78 (1H, d, J = 4.7Hz, C₅-H); 2.37 (3 H, s, CH₃). Data for 9: Mp 285° C. Analysis. Calcd.for C₂₁H₁₂F₃N₃O₄: C, 59.04: H, 2.80; N, 9.83 Found: C, 59.22; H, 2.94;N, 9.59 IR (KBr): 3178 (NH); 1733, 1664 (CO) cm⁻¹. ¹H-NMR (250 MHz,CDCl₃) d: 10.00 (1H, br, s, NH); 9.02 (1H, d, J = 4.8 Hz, C₇-H); 8.21(1H, s, NHCOCF₃); 7.70 (1H, d, J = 7.6 Hz, C_(6′)-H); 7.58 (1H, td, J =7.6 and 1.5 Hz, C_(4′)-H); 7.53 (1H, d, J = 4.8 Hz, C₆-H); 7.42 (1H, td,J = 7.6 and 1.5 Hz, C_(5′)-H); 7.16 (1H, dd, J = 7.6 and 1.5 Hz,C_(3′)-H); 6.46 (1H, d, J = 1.1 Hz, C₃-H); 2.46 (3 H, d, J = 1.1 Hz,CH₃).

[0044] Step 1(c) Oxidation of 7 to 9

[0045] To a suspension of compound 7 (R⁶=CH₃, R⁷=H), produced accordingto Step 1(b) above, [200 mg, 0.46 mmol in methanol (50 ml)] was addedsolid 10% palladium on charcoal (100 mg, 9 mmol). The suspension wasrefluxed for 48 h, while vigorously stirred, and filtered throughcelite. The solvent was evaporated, yielding 136 mg (69%) of the titlecompound 9 (R⁶=CH₃, R⁷=H).

[0046] Step 1(d)12-Methyl-9H-benzo[b]pyrido[4,3,2-d,e](1,7)phenantroline8,10-dione(IB-96213; compound of formula 10, R⁶=CH₃ and R⁷=H)

[0047] To a solution of compound 9 (R⁶=CH₃, R⁷=H) produced according toStep 1(d) above, in methanol (15 ml) was added 2 N aqueous hydrochloricacid (2 ml). The solution was refluxed for 4 h, neutralized with 8%aqueous sodium bicarbonate and extracted with chloroform. Evaporation ofthe organic layer gave 57 mg (78%) of the title compound 10 (R⁶=CH₃,R⁷=H). Data for 10: Mp > 300° C. (CHCl₃) Analysis. Calcd. forC₁₉H₁₁N₃O₂: C, 72.86: H, 3.51; N, 13.41 Found: C, 72.87; H, 2.82; N,13.57 IR (KBr): 3418 (NH); 1648 (CO and C = N) cm⁻¹. ¹H-NMR (250 MHz,F₃C-CO₂D) d: 9.24 (1H, br. s, C₆-H); 9.03 (1H, br. s, C₅-H); 8.57 (1H,d, J = 7.7 Hz, C₄-H); 8.22 (1H, d, J = 7.7 Hz, C₁-H); 7.96 (1H, t, J =7.3 Hz, C₂-H); 7.79 (1H, t, J = 7.3 Hz, C₃-H); 6.45 (s, 1H, C₁₁-H); 2.45(s, 3H, C₁₂-CH₃) ppm.

EXAMPLE 2

[0048] 9-Hydroxybenzo[b]prido[4,3,2-d,e](1,10)phenantrolin-8-one(IB-98205: a compound of formula 13 in which R⁷=R⁸=H)

[0049] Step 2(a) 4-Chloro-8-(o-nitrophenyl)-1,5-diazaanthraquinone (acompound of formula 12 in which R⁷=R⁸=H)

[0050] A solution of quinone 11 (see Scheme 4; R⁷=R⁸=H)¹⁵ (185 mg, 0.68mmol) and o-nitrocynammaldehyde dimethylhydrazone 1a¹² (438mg, 2 mmol)in chloroform (1 mL) was irradiated with ultrasound at 50° C. for 125 h.The solvent was evaporated and the residue was chromatographed on silicagel, eluting with ethyl acetate to give the title compound 11; yield, 52mg (20%). Data for 12: Mp, 146° C. Analysis. Calcd. for C₁₈H₈ClN₃O₄: C,59.13: H, 2.18; N, 11.48 Found: C, 59.89; H, 1.99; N, 11.17 IR (KBr)u:1689 (C = O) cm⁻¹. ¹H-NMR (250 MHz, CDCl₃) δ: 9.15 (d, 1H, J = 4.8 Hz,H-6); 8.81 (d, 1H, J = 5.1 Hz, H-2); 8.34 (dd, 1H, J = 7.6 and 1.3 Hz,H-3′); 7.77 (ddd, 1H, J = 7.6, 7.6 and 1.3 Hz, H-5′); 7.72 (d, 1H, J =5.1 Hz, H-3); 7.67 (ddd, 1H, J = 7.6, 7.6 and 1.3 Hz, H-4′); 7.49 (d,1H, J = 4.8 Hz, H-7); 7.28 (dd, 1H, J = 7.6 and 1.3 Hz, H-6′) ppm.¹³C-NMR (63 MHz, CDCl₃) δ: 180.66, 179.54, 155.03, 154.05, 150.36,149.86, 149.34, 146.91, 145.98, 134.15, 134.03, 131.06, 129.84, 129.59,128.44, 127.09, 126.61, 124.93, 121.46 ppm

[0051] Step 2(b) 9-Hydroxybenzo[b]pyrido[4,3,2-d,e](1,10)phenantrolin-8-one (13, R⁷=R⁸=H)

[0052] To a solution of compound 12 (100 mg, 0.27 mmol), producedaccording to Step 2(a) above, and triethylamine (37 mg) in methanol (25mL) was added 10% palladium on charcoal (22 mg). The suspension washydrogenated at 1 atm for 1 h and filtered through celite. The celitelayer was washed with a 2:1 mixture of trifluoroacetic acid andchloroform (25 mL). These washings were evaporated and chromatographedon silica gel, eluting with 9:1 ethyl acetate methanol, affording 45 mgof a mixture of two reduction intermediates, whose structure was notdetermined. To a solution of 35 mg of this mixture in methanol (20 mL)was added 10% palladium on charcoal (8 mg), and the suspension washydrogenated at 1 atm for 1 h and filtered through celite. The solutionwas evaporated and the residue was chromatographed on silica gel,yielding 6 mg (10%) of the title compound 13.

[0053]¹H-NMR (250 MHz, CF₃COOH) δ: 9.30(d); 9.15(d); 8.73(d); 8.36(m);8.10(m); 7.99(m); 7.48(d); 7.34(d) ppm.

[0054] The present inventors have prepared by the preceding syntheticpathway the following specific compounds, IB-96213 (general structure10, were R⁶=CH₃ and R⁷=H) and IB-98205 (general structure 13, wereR⁷=R⁸=H) which are specially preferred herein:

[0055] Biological Activity

[0056] The compounds of formula (I) of the present invention show goodantitumor activity. In particular, IB-96213 and IB-98205 display goodantitumor activity against several mammalian cancer cell lines. Itsantitumor activity has been detected in vitro by culturing the tumorcells following the methodology described by Bergeron et al. ¹⁶, and bySchroeder et al ¹⁶. Activity against different tumors as mouse lymphoma,human NSC lung carcinoma, human melanoma and human colon carcinoma hasbeen observed.

[0057] Some tumors were more sensitive than others. As for example itwas found that NSC lung carcinoma and melanoma cells were 100 times moresensitive than mouse lymphoma and 1000 times more sensitive than humancolon carcinoma cells.

EXAMPLE

[0058] Biological activity: Cells were maintained in logarithmic phaseof growth in Eagle's Minimum Essential Medium, with Earle's BalancedSalts, with 2.0 mM L-glutamine, with non-essential amino acids, withoutsodium bicarbonate (EMEM/neaa); suplemented with 10% Fetal Calf Serum(FCS), 10⁻² M sodium bicarbonate and 0,1 g/l penicillin-G+ streptomycinsulfate.

[0059] The tumor cell lines employed have been P-388 (ATCC CCL-46,suspension culture of a lymphoid neoplasm from DBA/2 mouse), A-549 (ATCCCCL-185, monolayer culture of a human lung carcinoma), HT-29 (ATCCHTB-38, monolayer culture of a human colon carcinoma) and MEL-28 (ATCCHTB-38, monolayer culture of a human melanoma).

[0060] P-388 cells were seeded into 16 mm wells at 1×10⁴ cells per wellin 1 ml aliquots of MEM 5FCS containing the indicated concentration ofdrug. A separate set of cultures without drug was seeded as controlgrowth to ensure that cells remained in exponential phase of growth. Alldeterminations were carried out in duplicate. After three days ofincubation at 37° C., 10% CO₂ in a 98% humide atmosphere, an aproximateIC₅₀ was determined by comparing the growth in wells with drug to thegrowth in wells control.

[0061] A-549, HT-29 and MEL-28 were seeded into 16 mm wells at 2×10⁴cells per well in 1 ml aliquots of MEM 10FCS containing the indicatedconcentration of drug. A separate set of cultures without drug wasseeded as control growth to ensure that cells remained in exponentialphase of growth. All determinations were carried out in duplicate. Afterthree days of incubation at 37° C., 10% CO₂ in a 98% humide atmosphere,the wells were stained with 0.1% Crystal Violet. An aproximate IC₅₀ wasdetermined by comparing the growth in wells with drug to the growth inwells control.

[0062] In Table I are presented the cytotoxicity expressed as IC₅₀ inμg/ml and μM TABLE I IC₅₀ μg/ml(μM) A-549 HT-29 MEL-28 P-388 ATCC- ATCCATCC COMPOUND ATCC CCL-46 CCL-185 HTB-38 HTB-38 IB-96213  0.1(0.32)0.001(0.003)   1(3.18) 0.001(0.003) IB-98205 1.25(4.18) 0.012(0.04) 0.12(0.40)  0.05(0.17) 

REFERENCES

[0063] 1. Reviews: a) T. F. Molinsky, Chem. Rev. 1993, 93, 1825. b) M.Alvarez, J. A. Joule, Heterocycles, 1992, 34, 2385. c) M. Alvarez, M.Salas, J. A. Joule, Heterocycles, 1991, 32, 759.

[0064] 2. J. Kobayashi, J. Cheng, M. R. Walchii, H. Nakamura, Y. Hirata,T. Sasaki, Y. Ohizumi, J. Am. Chem. Soc., 1988, 53, 1800.

[0065] 3. F. J. Schmitz, S. K. Agarwal, S. P. Gunasekera, J. Am. Chem.Soc., 1983, 105, 4835.

[0066] 4. F. J. Schmitz, F. S. de Guzman, M. B. Hossain, D. van derHelm, J. Org. Chem., 1991, 56, 804.

[0067] 5. N. Bontemps, I. Bonnard, B. Banaigs, G. Combaut, C. Francisco,Tetrahedron Lett., 1994, 35, 7023.

[0068] 6. J. Kobayashi, J.-F. Cheng, H. Nakamura, Y. Ohizumi, Y. Hirata,T. Sasaki, T. Ohta, S. Nozoe, Tetrahedron Lett., 1988, 29, 1177.

[0069] 7. 2-Bromoleptoclinidone: S. J. Bloor, F. J. Schmitz, TetrahedronLett., 1989, 30, 1069. Neocalliactine acetate: F. Bracher, Liebigs Ann.Chem., 1992, 1205.

[0070] 8. A. R. Carroll, P. J. Scheuer, J. Org. Chem., 1990, 55, 4426.

[0071] 9. N. M. Cooray, P. J. Scheuer, L Parkanyi, J. Clardy, J. Org.Chem., 1988, 53, 4619.

[0072] 10. A. Rudi, Y. Kashman, J. Org. Chem., 1989, 54, 5331.

[0073] 11. a) F. J. Schmitz, F. S. de Guzman, Y.-H. Choi, M. B. Hossain,S. K. Rizui, D. van der Helm, Pure Appl. Chem., 1990, 62, 1393. b) L. A.McDonald, G. S. Edredge, L. R. Barrows, C. M. Ireland, J. Med. Chem.,1994, 37, 3819. c) B. S. Lindsay, L. R. Barrows, B. R. Copp, Bioorg.Med. Chem. Lett., 1995, 5, 739.

[0074] 12. A. M. Echavarren, J. Org. Chem. 1990, 55, 4525.

[0075] 13. R. E. Dolle, W. P. Armstrong, A. N. Shaw, R. Novelli,Tetrahedron Lett., 1988, 29, 6349.

[0076] 14. The quinones used as dienophiles (compounds 6) were preparedfrom 2,5-dimethoxyaniline using previously published procedures: a) C.Avendaño, E. de la Cuesta, C. Gesto, Synthesis, 1991, 727. b) M. M.Blanco, C. Avendaño, N. Cabezas, J. C. Menéndez, Heterocycles, 1993, 36,1387. c) M. A. Alonso, M. M. Blanco, C. Avendaño, J. C. Menéndez,Heterocycles, 1993, 36, 2315. d) L. M. Diaz-Guerra, B. Ocaña, J. M.Pérez, C. Avendaño, M. Espada, J. C. Menéndez, M. T. Ramos, M. A. Ruiz,J. M. Pingarrón, D. Salvatierra, C. Jaime, Bull. Soc. Chim. Belg., 1995,104, 683. e) P. López-Alvarado, C. Avendaño, J. C. Menéndez, Synthesis,1998, 186.

[0077] 15. Gómez-Bengoa, E.; Echavarren, A.M., J. Org. Chem., 1991, 56,3497.

[0078] 16. Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline,Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter. Antineoplasticand antiherpetic activity of spermidine catecholamide iron chelators.Biochem. Bioph. Res. Comm. 1984, 121(3): 848-854.

[0079] 17. Alan C. Schroeder, Robert G. Hughes, Jr. and Alexander Bloch.Effects of Acyclic Pyrimidine Nucleoside Analoges. J. Med. Chem. 1981,24:1078-1083.

1. A compound of formula (I):

wherein: X is selected from the group consisting of O, and NR³, where R³represents a lower alkyl group; Y is selected from the group consistingof CH and N; R¹ and R² are independently selected from the groupconsisting of NH₂, NHR⁴ and NR⁵ ₂, where R⁴ and R⁵ each represent alower alkyl group, or R¹ and R² together represent a cycle selected from(a), (b) and (c):

wherein R⁶, R⁷ and R⁸ are independently selected from the groupconsisting of hydrogen atoms, lower alkyl groups, hydroxy groups andlower alkoxy groups; and Z is selected from the group consisting of Oand NH; and pharmaceutically acceptable salts thereof.
 2. A compoundaccording to claim 1, wherein X represent an oxygen atom.
 3. A compoundaccording to claim 1 or claim 2, wherein Y represent a nitrogen atom. 4.A compound according to any one of claims 1 to 3, wherein R¹ and R²together represent a cycle of formula (a).
 5. A compound according toany one of claims 1 to 3, wherein R¹ and R² together represent a cycleof formula (b).
 6. A compound according to any one of claims 1 to 3,wherein R¹ and R² together represent a cycle of formula (c).
 7. Acompound according to claim 4, wherein R⁶ and R⁷ are independentlyselected from the group consisting of hydrogen atoms, alkyl groupshaving from 1 to 4 carbon atoms, hydroxy groups and alkoxy groups havingfrom 1 to 4 carbon atoms.
 8. A compound according to claim 7, wherein R⁶and R⁷ are independently selected from the group consisting of hydrogenatoms, methyl groups and ethyl groups.
 9. A compound according to claim8, wherein R⁶ represents a methyl group and R⁷ represents a hydrogenatom.
 10. A compound according to any one of claims 4 and 7 to 10,wherein Z represents a group of formula NH.
 11. A compound according toclaim 6, wherein R⁶, R⁷ and R⁸ are selected from the group consisting ofhydrogen atoms, alkyl groups having from 1 to 4 carbon atoms, hydroxygroups and alkoxy groups having from 1 to 4 carbon atoms.
 12. A compoundaccording to claim 11, wherein R⁶, R⁷ and R⁸ are independently selectedfrom the group consisting of hydrogen atoms, methyl groups, ethyl groupsand hydroxy groups.
 13. A compound according to claim 12, wherein R⁶represents a hydroxy group and R⁷ and R⁸ each represent a hydrogen atom.14. A compound according to claim 1 having the following formula (II):

wherein R⁶ and R⁷ are as defined in claim
 1. 15. A compound according toclaim 14, wherein R⁶ represents a methyl group and R⁷ represents ahydrogen atom.
 16. A compound according to claim 1 having the followingformula (III):

wherein R⁷ and R⁸ are as defined in claim
 1. 17. A compound according toclaim 16, wherein R⁷ and R⁸ each represent a hydrogen atom.
 18. Apharmaceutical composition comprising a compound of formula (I) asdefined in any one of claim 1 to 17 in admixture with a pharmaceuticallyacceptable carrier or diluent.
 19. A pharmaceutical compositionaccording to claim 18 having antitumor activity.
 20. A pharmaceuticalcomposition according to claim 19, for the treatment of malignanttumors.
 21. A compound of formula (I) according to any one of claims 1to 17 for use in therapy.
 22. Use of a compound of formula (I) accordingto any one of claims 1 to 17 in the manufacture of a medicament for thetreatment of malignant tumors.
 23. A method for treating a mammalaffected by a maligant tumor, comprising the administration to theaffected individual of an effective amount of a compound of formula (I)according to any one of claims 1 to
 17. 24. A process for the productionof compounds of formula (II), as defined in claim 14, comprising: (a)reaction of a compound of formula 1b:

with a compound of formula 6:

wherein R⁶ and R⁷ are as defined in claim 1, to give a compound offormula 7:

wherein R⁶ and R⁷ are as defined in claim 1; (b) aromatization of thecompound of formula 7 in the presence of a Pd-C catalyst, to give acompound of formula 9:

wherein R⁶ and R⁷ are as defined in claim 1; and (iii) hydrolyticcyclization of the compound of formula 9 to give the compound of formula(II).
 25. A process for the production of a compound of formula (III),as defined in claim 16, comprising: (a) reaction of a compound offormula 1a:

with a compound of formula 11:

wherein R⁷ and R⁸ are as defined in claim 1, to give a compound offormula 12:

wherein R⁷ and R⁸ are as defined in claim 1: and (b) hydrogenation ofthe compound of formula 12 in the presence of a Pd-C catalyst followedby acidic hydrolytic work-up, to give the compound of formula (III).